This invention relates to a dry stain which can be mixed with whole blood to prepare a microscope slide, and to a method of preparing the stain.
The determination of reticulocyte count as a percentage of total red cells is an extremely useful and important procedure in clinical hematology. A number of techniques have been used for staining blood for such analysis. Most of these techniques involve the use of a liquid stain preparation which is mixed with the blood.
Three techniques for reticulocyte staining are described in Staining Procedures (3rd Ed.) edited by George Clark, published for the Biological Stain Commission by the Williams and Wilkens Company, Baltimore, Maryland (pp. 128-130). These techniques are described in the following publications: (1) Brecher, G., New Methylene Blue as a Reticulocyte Stain, American Journal of Clinical Pathology, Vol. 19, pp. 895-896 (1949), (2) Robertson, O. H., The Effects of Experimental Plethora on Blood Production, Journal of Experimental Medicine, Vol. 26, pp. 221-237 (1917), and (3) Cunningham, R. S., A Method for Permanent Staining of Reticulated Red Cells, Archives of Internal Medicine, Vol. 26, pp. 405-409 (1920).
The technique described by Brecher uses a solution of 0.5% new methylene blue N and 1.6% potassium oxalate in water, and this solution is mixed with blood in equal volumes. After 10 to 15 minutes a drop is placed on a slide and a smear is made in a normal manner. The Brecher formula, which is presently the most widely used, has been favored because of the sharper reticulum it produces and because of the consistency of dye purity from batch to batch. One of the problems associated with the Brecher stain is that precipitate forms in it continuously over a long period of time, and therefore, in order to avoid slide artifacts which might interfere with the count, the stain must be filtered each time it is used. If the stain is dried, precipitates form when it is redissolved. Even if the insoluble matter is removed directly prior to drying, more precipitate forms immediately when the stain is dissolved in the blood. This precipitate is probably an insoluble salt of the divalent anions and the new methylene blue N dye.
The Robertson publication describes a wet procedure using brilliant cresyl blue. A saturated aqueous stock solution of the stain is prepared in 0.85% NaCl. The stock is diluted 80-180 times, and 20 volumes of the dilute solution are mixed with one volume of blood. The counts are made of fresh preparations sealed with vasoline to prevent drying. The technique reported by Cunningham also uses brilliant cresyl blue followed by Wright's stain. Using a solution of 0.3% alcoholic brilliant cresyl blue, a drop is dried on a slide. A drop of blood is placed on the stain, mixed, spread and dried. The smear is then counterstained by the normal technique with Wright's stain.
One of the problems with liquid stains is that insoluble granules form continuously in the solution. Since such a precipitate can produce artifacts on the slide which would interfere with the reticulocyte count, the stain must be filtered each time it is used to remove this residue. Following filtration, the stain must be volumetrically dispensed into the blood that is to be analyzed. These are time consuming, incovenient and messy operations for the modern clinical laboratory.